EAGER: Collaborative Research: Tracking of KOR1 Protein Transport in Arabidopsis using Fluorescent-Timer Imaging System

EAGER：合作研究：使用荧光定时器成像系统追踪拟南芥中的 KOR1 蛋白转运

• 批准号: 1547551
• 负责人: Hisashi Koiwa
• 金额: $ 25.25万
• 依托单位: Texas A&M AgriLife Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1547551&HistoricalAwards=false
• 关键词: EAGER; Collaborative; Research; Tracking; KOR1

In this project, methods will be developed to allow direct observation of the movement of a protein, KORRIGAN1 (KOR1), in plant cells. KOR1 functions in the formation of cellulose fibers, which are the primary constituents of plant cell walls. Cellulose fiber deposition that is controlled by KOR1 influences cell growth and biomass production. KOR1 travels from one compartment to another inside cells before it finally reaches the cell surface where cellulose fibers will ultimately be deposited. To visualize the dynamics of KOR1 in plant cells, this project will apply fluorescent-timer (FT) technology, in which each KOR1 protein is labeled with a time-sensitive fluorescent tag. Because FT changes its color over time, fluorescence images of cells expressing FT-tagged KOR1 will allow visualization of the age and location of KOR1 inside of the cells. This will reveal the route of KOR1 transport through the plant cell. By observing how KOR1 transport changes under different biological circumstances, these studies will reveal how certain factors influence the paths that proteins travel within cells. This is a novel research tool/approach in plant biology that can change the way the scientists view proteins in any plant cell.In plants, distribution of KOR1 among different subcellular domains, such as trans-Golgi network (TGN), plasmamembrane (PM), tonoplast (TP), as well as the cell plate in case of the dividing cells, is scrupulously maintained. Dysfunction of KOR1 is often associated with increased targeting to TP. This project aims to visualize the dynamics of KOR1 transport beyond analysis of endpoint locations. Two transport processes relevant to KOR1 biology will be studied. First, transport route/kinetics and a redirection mechanism of de-novo-synthesized KOR1 will be analyzed by tracking the age of KOR1 after induction of protein expression. Next, KOR1 age information will be used to dissect the multiple targeting routes of KOR1 to the cell plate during cell division. These systems will serve as models for further dissection of various factors that quantitatively affect KOR1 transport. The interdisciplinary team will optimize FT technology for plant cell biology as a whole fluorescence analysis package, including FT with characterized in planta maturation time, compatible fluorescent subcellular markers, and automated image-processing pipelines. The experimental platform established in this project will serve as the basis for further investigation of multiple factors that affect KOR1 transport, including protein N-glycosylation pathways and interactions with cellulose synthase complex.

在该项目中，将开发能够直接观察植物细胞中蛋白质 KORRIGAN1 (KOR1) 运动的方法。 KOR1 在纤维素纤维的形成中发挥作用，纤维素纤维是植物细胞壁的主要成分。 KOR1 控制的纤维素纤维沉积影响细胞生长和生物量生产。 KOR1 从细胞内的一个室移动到另一个室，然后最终到达细胞表面，纤维素纤维最终沉积在细胞表面。 为了可视化 KOR1 在植物细胞中的动态，该项目将应用荧光计时器 (FT) 技术，其中每个 KOR1 蛋白都用时间敏感的荧光标签进行标记。 由于 FT 的颜色会随着时间的推移而改变，因此表达 FT 标记的 KOR1 的细胞的荧光图像将允许可视化 KOR1 在细胞内的年龄和位置。 这将揭示 KOR1 通过植物细胞的运输路线。 通过观察 KOR1 转运在不同生物环境下的变化，这些研究将揭示某些因素如何影响蛋白质在细胞内的移动路径。 这是植物生物学中的一种新颖的研究工具/方法，可以改变科学家看待任何植物细胞中蛋白质的方式。在植物中，KOR1 在不同亚细胞结构域（例如反高尔基体网络 (TGN)、质膜 (PM)、液泡膜 (TP) 以及分裂细胞的细胞板）之间的分布得到严格维护。 KOR1 的功能障碍通常与 TP 靶向性增加有关。 该项目的目的是在端点位置分析之外可视化 KOR1 传输的动态。 将研究与 KOR1 生物学相关的两个运输过程。 首先，将通过跟踪诱导蛋白表达后 KOR1 的年龄来分析从头合成的 KOR1 的运输路线/动力学和重定向机制。 接下来，KOR1年龄信息将用于剖析细胞分裂过程中KOR1到细胞板的多种靶向途径。 这些系统将作为进一步剖析定量影响 KOR1 运输的各种因素的模型。 该跨学科团队将优化植物细胞生物学的 FT 技术作为一个完整的荧光分析包，包括以植物成熟时间为特征的 FT、兼容的荧光亚细胞标记和自动图像处理流程。本项目建立的实验平台将作为进一步研究影响KOR1转运的多种因素的基础，包括蛋白质N-糖基化途径以及与纤维素合酶复合物的相互作用。